Detection, analysis and quantification of an analyte, such as a cell, macromolecule, protein, polymer, biomolecule, biopolymer or other molecular complex has widespread application in fields such as genomics, proteomics, drug discovery, medical diagnostics, environmental sensing, pollution monitoring, detection of chemical or biological warfare agents, and industrial process monitoring. For example, cell and tissue-based biosensors are useful for many medical, pharmaceutical, and environmental applications. One significant advantage of using living elements in sensors is that, in principle, complex biological interactions can be monitored. However, many biological events that signal changes in cellular physiology cannot readily be converted into an electronic signal in real-time.
Chemical reactions can signal the occurrence of many events of interest. The reaction activity of enzymes and otherwise catalytic or reactive molecules signals many important biological and non-biological reactions, for example. Such reactions are of interest to researchers to understand and quantify many biological, genetic, and general chemical processes. Research in many fields, including medical, biological, military, and industrial fields seeks to better understand, recognize and quantify the activity of enzymes or otherwise catalytic or reactive molecules.
Protease research is one such field. Proteases are enzymes that break peptide bonds between amino acids of proteins. The activity of proteases is an indicator of many biological processes. Protease activity is therefore a widespread area of research. Protease activity can quickly degrade cells, and is regulated by protease inhibitors. Promoting protease activity to target certain cells, e.g., tumor cells, is one area of research. Inhibiting protease activity to prevent harmful cell destruction such as in HIV research is another area of intense research. As proteases are included in every living cell, the conditional activation and inhibition of protease activity holds great promise in many areas of biological and genetic research.
There is a need for quick assays of protease activity because of their pivotal intra- and inter-cellular role in biological systems. Schemes to quantify protease activity primarily involve either fluorescent or colorimetric assays that are time-consuming and that require relatively large quantities of enzyme. In addition, these assays use substrates that have been modified from their native forms in order to incorporate the relevant indicator chemistries. A typical colorimetric assay, such as from Athena Enzyme systems, requires incubation times up to twenty four hours.
A standard method to detect protease activity uses a fluorescent molecule conjugated to casein or bovine serum albumin substrates. Some concerns with using fluorescent-conjugated substrates are that the presence of the dye may affect the proteolytic cleavage rate, quantification requires a sensitive fluorimeter, and the reagents are costly. The well-known colorimetric assay using the Folin-Ciocalteu reagent operates on native substrates, but it is less sensitive than fluorescence methods and requires an extensive workup procedure.
Research efforts and progress are slowed by such typical conventional methods for detecting protease activity. Relatively large quantities of time and reagents are required, costs can be high, and procedures can be complicated.
The intensity of a porous thin film's visible photoluminescence, e.g., porous silicon, changes depending upon the types of gases absorbed to its surface. This phenomenon constitutes the basis for a simple and inexpensive chemical sensor device. See, U.S. Pat. No. 5,338,415. Porous thin films, e.g., porous silicon, insulator and semiconductor films, can be fabricated to display well-resolved Fabry-Perot fringes in their luminescence and reflection spectra. Such interference-based spectra are sensitive to gases or liquids adsorbed to the inner surfaces of the porous Si layer. See, U.S. Pat. No. 5,318,676, which uses the interference based spectra to identify adsorbed individual gases or liquids. See also, U.S. Pat. No. 6,248,539 used a binder to bind analytes in pores and then identify the analytes by detecting a shift in the reflection spectra. Porous films and particles for sensing are also discussed in U.S. Published Application No. 20060255008, published Nov. 16, 2006, and entitled Photonic Sensor Particles and Fabrication Methods, which discloses use of optical particles in sensing applications, and methods of fabricating optical particles that can target a desired analyte. Strategies for encoding porous films are also discussed in the following U.S. Published Applications: 20060236436, published Oct. 19, 2006 and entitled Nanostructured Casting of Organic and Bio-Polymers in Porous Silicon Templates; 20060105043, published May 18, 2006 and entitled Porous Nanostructures and Methods involving the same; 20050042764, published Feb. 24, 2005 and entitled Optically Encoded Particles; 20050009374, published Jan. 13, 2005 and entitled Direct Patterning of Silicon by Photoelectrochemical Etching; 20040152135, published Aug. 4, 2004 and entitled Porous Semiconductor-Based Optical Interferometric Sensor; and 20030146109, published Aug. 7, 2003 and entitled Porous thin film time-varying reflectivity analysis of samples. Also see U.S. Pat. Nos. 6,897,965, 6,720,177, and 6,248,539, which disclose semiconductor based optical interferometric sensors and discuss fabrication of the same.